WO2009131179A1 - Yeast mutant and substance production process using the same - Google Patents

Abstract

The object aims to largely improve the productivity of a desired product and maintain an excellent growth rate and an excellent fermentation rate in the production of the desired product by using yeast. A foreign gene which encodes an enzyme involved in the production of the desired product and HAP4 gene which can be expressed constitutively or a homologous gene thereof are introduced into a yeast host. The yeast mutant is preferably a mutant strain having reduced alcohol productivity compared to its wild-type one.

Description

Mutant yeast and substance production method using the same

The present invention relates to a mutant yeast modified so that a predetermined gene is constitutively expressed in wild type yeast, and a substance production method using the same.

When producing a target product using yeast (Saccharomyces cerevisiae), etc., a mutant yeast introduced so that the gene involved in biosynthesis of the target product can be expressed constitutively is prepared and subjected to appropriate culture conditions. The mutant yeast is then cultured, and the target product is recovered from inside or outside the fungus body. When a substance other than ethanol is used as the target product, it is preferable to reduce ethanol produced in large quantities. So far, gene-disrupted strains such as pyruvate decarboxylase and alcohol dehydrogenase, which are in the ethanol production pathway, have been produced in order to reduce ethanol production ability. However, especially in bacteria having a Crabtree effect such as Saccharomyces cerevisiae, although the ethanol production amount is reduced, there is a problem that the growth and fermentation ability are greatly reduced and the practicality is low. (Non-patent document 1: Ishida, N. et al. (2006) Biosci. Biotechnol. Biochem. 70, p1148-1153, Non-patent document 2: Flikweert, MT et al. (1996) Yeast 12, p247-257, non Patent Document 3: Eri, A. et al. (1998) J. Ferment. Bioeng. 86, p284-289, Patent Literature 1: Special Table 2003-500062, Patent Literature 2: Special Table 2001-516584, Non-patent document 4: Skory, CD (2003) J. Ind. Microbiol. Biotechnol 30, p22-27). Further, according to Non-Patent Document 1, Non-Patent Document 3 and Non-Patent Document 4, even though ethanol can be significantly reduced by destroying a gene in the ethanol production pathway, the majority of the reduction is not necessarily made. It is not a target product.

On the other hand, there is a report that the production pathway of ethanol is only partially blocked and the metabolic pathway of the target product is enhanced to improve the target product yield (Non-patent Document 5: Saitoh, S (2005)). Appl. Environ. Microbiol. 71, p2789-2792) Although the yield of the target product is improved, there is a problem that ethanol reduction is insufficient and the fermentation rate is slightly reduced.
Special table 2003-500062 Special table 2001-516584 Ishida, N. et al. (2006) Biosci. Biotechnol. Biochem. 70, p1148-1153 Flikweert, MT et al. (1996) Yeast 12, p247-257 Eri, A. et al. (1998) J. Ferment. Bioeng. 86, p284-289 Skory, CD (2003) J. Ind. Microbiol. Biotechnol 30, p22-27 Saitoh, S (2005) Appl. Environ. Microbiol. 71, p2789-2792

Therefore, in view of the above situation, the present invention can significantly improve the productivity of the target product and maintain an excellent growth rate and fermentation rate when producing the target product using yeast. An object of the present invention is to provide a mutant yeast that can be produced and a method for producing a substance using the same.

As a result of intensive studies by the present inventors in order to achieve the above-mentioned object, the growth rate and fermentation rate are maintained by introducing a gene involved in the production of the target product and constitutively expressing the HAP4 gene. However, it has been found that the productivity of the target product is greatly improved, and the present invention has been completed.

The present invention includes the following.

(1) A mutant yeast introduced with a foreign gene encoding an enzyme involved in the production of a target product and a constitutively expressible HAP4 gene or a homologous gene thereof.

The mutant yeast of (1) above is preferably a mutant strain having reduced alcohol productivity as compared to the wild type. For example, alcohol productivity can be reduced by reducing the enzyme activity of an enzyme involved in alcohol synthesis. Here, examples of the enzyme involved in alcohol synthesis include pyruvate decarboxylase and / or alcohol dehydrogenase. Examples of the pyruvate decarboxylase include an enzyme encoded by at least one gene selected from the group consisting of PDC1 gene, PDC5 gene and PDC6 gene. Examples of the alcohol dehydrogenase include an enzyme encoded by the ADH1 gene. Moreover, as the mutant yeast of (1) above, it is preferable to use yeast belonging to the genus Saccharomyces, particularly yeast belonging to the strain of Saccharomyces cerevisiae. Examples of the foreign gene include a gene encoding a protein having lactate dehydrogenase activity.

(2) A substance production method using yeast, comprising the steps of culturing the mutant yeast according to the present invention described above to produce the target product inside and outside the fungus body and the step of recovering the target product.

In the substance production method of (2) above, the target product can be an organic acid. In particular, the target product is preferably lactic acid. The target product may be alcohol other than ethanol.

According to the present invention, by using the mutant yeast according to the present invention, which can provide a mutant yeast excellent in the productivity of the target product while maintaining an excellent growth rate and fermentation rate, The target product can be produced at a low cost.

This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2008-113053 which is the basis of the priority of the present application.

It is a schematic diagram which shows the flow which constructs | assembles the DNA fragment for LDH gene transfer / PDC1 gene destruction. It is a schematic diagram which shows the flow which constructs | assembles the DNA fragment for PDC5 gene destruction. It is a schematic diagram showing a flow for constructing a DNA fragment for HAP4 gene overexpression. It is a characteristic view which shows the result of a fermentation test about the HAP4 introduction | transduction strain | stump | stock and HAP4 non-introduction strain | stump | stock with respect to a LDH gene introduction | transduction / PDC1 gene destruction strain. It is a characteristic view which shows the result of a fermentation test about HAP4 introduction | transduction strain | stump | stock and HAP4 non-introduction strain | stump | stock with respect to a LDH gene introduction | transduction / PDC1 and PDC5 gene disruption strain.

Hereinafter, the present invention will be described in detail.

The mutant yeast according to the present invention is obtained by introducing a foreign gene encoding an enzyme involved in production of a target product and a constitutively expressible HAP4 gene or a homologous gene thereof. Here, the HAP4 gene encodes the HAP4 protein constituting the subunit of the Hap2p / 3p / 4p / 5p CCAAT-binding complex that is hemi-activated and glucose-repressed. The complex is known to have transcription promoting activity for various genes. In particular, the HAP4 protein and the above-mentioned complex are described in detail in Gancedo (JM (1998) Yeast carbon catabolite repression. Microbiol. Mol. Biol. Rev. 62 (2), p334-61.

The nucleotide sequence of the coding region in the HAP4 gene and the amino acid sequence of the HAP4 protein are shown in SEQ ID NOs: 1 and 2, respectively. The HAP4 gene to be introduced so as to be constitutively expressed is not limited to the protein containing the amino acid sequence shown in SEQ ID NO: 2, and is preferably 70% or more, for example, with respect to the amino acid sequence shown in SEQ ID NO: 2. Contains an amino acid sequence having an identity of 80% or more, more preferably 90% or more, more preferably 95% or more, and most preferably 97% or more, and encodes a protein that forms a complex and exhibits transcription promoting activity It may be a gene. Here, the identity means a value obtained by default setting using a computer program in which the blast algorithm is implemented and a database storing gene sequence information.

In addition, the HAP4 gene may be one or more (for example, 2 to 60, preferably 2 to 50, more preferably 2 to 40, still more preferably 2 to 30) in the amino acid sequence shown in SEQ ID NO: 2. Most preferably, it may be a gene encoding a protein which comprises an amino acid sequence in which 2 to 15 amino acids have been deleted, substituted, added or inserted and which forms the above-mentioned complex and exhibits transcription promoting activity.

Furthermore, the HAP4 gene is not limited to the gene containing the base sequence shown in SEQ ID NO: 1, but for all or a part of a continuous polynucleotide comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1. Alternatively, it may be a gene that encodes a protein that hybridizes under stringent conditions to form the complex and exhibits transcription promoting activity. Here, stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, hybridization at 45 ° C. and 6 × SSC (sodium chloride / sodium citrate), followed by washing at 50 to 65 ° C., 0.2 to 1 × SSC, 0.1% SDS, or such conditions , 65-70 ° C., hybridization at 1 × SSC, and subsequent washing at 65-70 ° C., 0.3 × SSC. Hybridization can be performed by a conventionally known method such as the method described in J. Sambrook et al. Molecular lonCloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).

The amino acid sequence having a predetermined identity as described above, the amino acid sequence having an amino acid deletion, substitution or addition, etc., is a base sequence encoding a protein containing the amino acid sequence shown in SEQ ID NO: 2 (for example, And the polynucleotide having the base sequence shown in SEQ ID NO: 1 can be modified by a technique known in the art. Similarly, a polynucleotide that hybridizes under stringent conditions to all or a part of a polynucleotide consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 similarly to SEQ ID NO: The polynucleotide having the base sequence shown in 1 can be modified by a technique known in the art. Mutation can be introduced into a nucleotide sequence by a known method such as Kunkel method or Gapped duplex method or a method according thereto, for example, a mutation introduction kit using site-directed mutagenesis (for example, Mutant- Mutations are introduced using K, Mutant-G (both trade names, manufactured by TAKARA Bio Inc.) or the like, or using LA PCR-in-vitro Mutagenesis series kits (trade name, manufactured by TAKARA Bio Inc.). In addition, as a method for introducing mutations, EMS (ethyl methanesulfonic acid), 5-bromouracil, 2-aminopurine, hydroxylamine, N-methyl-N'-nitro-N nitrosoguanidine, and other carcinogenic compounds are representative. A method using a chemical mutagen such as that described above may be used, or a method using radiation treatment or ultraviolet treatment represented by X-rays, alpha rays, beta rays, gamma rays and ion beams may be used.

In addition, whether a certain protein forms the above-mentioned complex and has a transcription promoting activity is determined by, for example, David S. ら McNabb et al., Eukaryotic Cell, November 2005, p. 1829-1839, Vol. 4, No. 11 This can be confirmed using the experimental system disclosed in (1). That is, this document describes that a Hap4 protein is allowed to act after constructing a Hap2p / Hap3p / Hap5p heterotrimer, thereby forming the complex and exhibiting the transcription promoting activity. Therefore, it is possible to easily examine whether or not a certain protein has a function equivalent to that of the HAP4 protein using the experimental system described in this document.

By the way, the above-mentioned HAP4 gene can be isolated from Saccharomyces cerevisiae by a conventionally known technique and used. In the present invention, a homologous gene of the HAP4 gene may be used in place of the above-described HAP4 gene. HAP4 gene homologous genes include Kluyveromyces lactis-derived HAP4 homologous genes (see Bourgarel, D. et al., 1999. Mol. Microbiol. 31: 1205-1215) and Hansenula polymorpha-derived HAP4 homologous genes (Sybirna, K. et. al., 2005. Curr. Genet. 47: 172-181). The base sequences of these HAP4 homologous genes and the amino acid sequences of HAP4 homologous proteins encoded by the genes can be obtained from known databases such as Genbank.

In order to constitutively express the HAP4 gene or its homologous gene described above in the host, for example, a method using a constitutive expression promoter can be mentioned. That is, a method of constructing an expression vector in which the HAP4 gene or a homologous gene thereof is arranged under the control of a constitutive expression promoter and transforming a host using the expression vector can be employed. Here, the constitutive expression promoter is a promoter having a function of expressing a downstream gene regardless of the growth conditions of the host cell. The constitutive expression promoter can be used without particular limitation, but may be appropriately selected according to the type of host cell and the type of gene to be controlled. For example, constitutive expression promoters in Saccharomyces cerevisiae include ADH1 promoter, HIS3 promoter, TDH3 promoter, CYC3 promoter, CUP1 promoter and HOR7 promoter.

The expression vector containing the above-described constitutive expression promoter and the HAP4 gene or a homologous gene thereof is another sequence for preparing the expression of the HAP4 gene or the homologous gene when introduced into a host, specifically an operator. , Enhancer, silencer, ribosome binding sequence, terminator and the like.

Here, the host for introducing the HAP4 gene or its homologous gene so as to constitutively express it is not particularly limited as long as it is a yeast. Examples of yeasts that can be used as a host include Ascomycota ascomycete yeast, Basidiomycota basidiomycetous yeast, and incomplete fungal (Fungi Imperfecti) incomplete fungal yeast. . Preferably, ascomycetous yeast, particularly Saccharomyces cerevisiae, budding yeast, Candida utilis or Pichia pastris, Shizosaccharomyces pombe, etc. Used. Moreover, Kluyveromyces lactis and Hansenula polymorpha can be used as hosts.

In particular, for the yeast used as a host, it is preferable to use a mutant having reduced alcohol productivity. Here, that alcohol productivity fell means that alcohol productivity fell significantly compared with wild type yeast. For example, alcohol productivity can be reduced by introducing a mutation into wild-type yeast so that the enzyme activity of an enzyme involved in alcohol synthesis is reduced. Examples of enzymes involved in alcohol synthesis include pyruvate decarboxylase and alcohol dehydrogenase. Among these pyruvate decarboxylase and alcohol dehydrogenase, alcohol productivity can be reduced by reducing one or both enzyme activities. Examples of the gene encoding pyruvate decarboxylase derived from Saccharomyces cerevisiae include PDC1 gene, PDC5 gene and PDC6 gene. Examples of the gene encoding alcohol dehydrogenase derived from Saccharomyces cerevisiae include the ADH1 gene.

Alcohol productivity can be reduced by deleting one or more of these genes. The method for deleting the gene is not particularly limited, but includes a method for deleting the gene, a method for introducing a mutation into the gene to express an inactive enzyme, and an expression control region of the gene (for example, a promoter). The method of deleting or mutating can be mentioned. Further, the gene deletion method includes a method of expressing siRNA (small interfering RNA), antisense RNA, and ribozyme for the gene in a host cell.

The mutant yeast according to the present invention is a foreign gene encoding an enzyme involved in the production of the target product, and can be used to produce the target product. The target product is not particularly limited as long as it is a substance that can be biosynthesized in yeast. For example, lactic acid, acrylic acid, acetic acid, pyruvic acid, 3-hydroxypropionic acid, fumaric acid, succinic acid, itaconic acid, levulinic acid, adipine Examples include acids, organic acids such as ascorbic acid and citric acid, and alcohols such as 1-propanol, 2-propanol, 1-butanol, isobutanol, 2-methyl-1-butanol and 3-methyl-1-butanol. .

In particular, when lactic acid is the target product, examples of the foreign gene include a lactate dehydrogenase (LDH) gene involved in lactic acid synthesis. In other words, by introducing the LDH gene as a foreign gene, the mutant yeast is imparted with lactic acid-producing ability. LDH has various homologues depending on the type of organism or in vivo. The LDH used in the present invention includes not only naturally derived LDH but also LDH synthesized chemically or genetically. LDH is preferably derived from prokaryotes such as Lactobacillus helvetics, Lactobacillus casei, Klubermyces thermotolerance, Torlas pora del brucchi, Schizosaccharomyces pombe, Rhizopus oryzae and other eukaryotes such as mold More preferably, it is derived from higher eukaryotes such as plants, animals and insects. For example, it is preferable to use bovine-derived LDH (L-LDH). By introducing these genes into the yeast described above, the microorganism can be imparted with lactic acid-producing ability.

Further, the above-described foreign gene may be introduced under the control of a constitutive expression promoter, or may be introduced under the control of an expression-inducible promoter. The foreign gene described above need not be introduced under the control of a promoter containing a CCAAT consensus sequence that is recognized and bound by the Hap2p / 3p / 4p / 5p CCAAT-binding complex.

According to the mutant yeast according to the present invention, the HAP4 gene or a homologous gene thereof is constitutively expressed, so that the productivity of the target product is greatly improved. In particular, in mutant yeast with reduced alcohol productivity, constitutive expression of the HAP4 gene or its homologous gene improves the productivity of the target product without reducing the growth rate and fermentation rate. be able to. Thus, by using the mutant yeast according to the present invention, the yield of the target product can be greatly improved, and the production cost of the target product can be significantly reduced.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the technical scope of the present invention is not limited to the following examples.

[Example 1]
LDH gene transfer / PDC1 gene disruption DNA fragment preparation First, the lactate dehydrogenase (LDH) gene is introduced into the host yeast as a foreign gene, and the pyruvate synthase gene (PDC1 gene involved in alcohol synthesis) A DNA fragment was prepared to break down.

Specifically, a DNA fragment fused with PDC1 promoter, LDH gene and TDH3 terminator was amplified by PCR using plasmid pBTrp-PDC1-LDHKCB disclosed in JP-A-2003-259878 as a template. In this PCR, TB215 (5′-GAAACAGCTATGACCATGATTACG-3 ′: SEQ ID NO: 3) and TB1497 (5′-AAGCTCTTAAAACGGGAATTCCCCTAAGAAACCAT-3 ′: SEQ ID NO: 4) were used as primers (see FIG. 1).

Meanwhile, the HIS3 gene was amplified using plasmid pRS403 (available from ATCC) as a template. In this PCR, TB1421 (5′-ATGGTTTCTTAGGGGAATTCCCGTTTTAAGAGCTT-3 ′: SEQ ID NO: 5) and TB422 (5′-GACCAAGTTAGCTGGTCGAGTTCAAGAGAAAAAAAAAG-3 ′: SEQ ID NO: 6) were used as primers.

In addition, the downstream DNA fragment of the PDC1 gene was amplified by PCR using the above pBTrp-PDC1-LDHKCB as a template. In this PCR, TB1147 (5′-CCAGCTAACTTGGTCGACTTG-3 ′: SEQ ID NO: 7) and TB019 (5′-GCGCGTAATACGACTCACTAT-3 ′: SEQ ID NO: 8) were used as primers.

Using the three PCR products amplified as described above as templates, the method of Shevchuk, NA et al. (Shevchuk, N. A. et al. (2004) Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously. Three kinds of PCR products were combined by Nucleic® Acids® Research® 32 (2) e19). In this PCR, TB151 (5′-CCTATCTCTAAACTTCAACACC-3 ′: SEQ ID NO: 9) and TB152 (5′-TCAGCAATAGTGGTCAACAACT-3 ′: SEQ ID NO: 10) were used as primers. The obtained DNA fragment contains a region where the PDC1 promoter, LDH gene and TDH3 terminator are fused in this order, the LEU2 gene as a selection marker, and the downstream region of the PDC1 gene as a recombination region. . Hereinafter, this DNA fragment is referred to as “LDH gene introduced / PDC1 gene disruption DNA fragment”.

Preparation of DNA fragment for PDC5 gene disruption In this example, a DNA fragment for disrupting the PDC5 gene was prepared. Specifically, using the genomic DNA of Saccharomyces cerevisiae BY4742 (Invitrogen) as a template, the 5 ′ upstream untranslated region DNA fragment and the 3 ′ downstream untranslated region DNA fragment of the PDC5 gene were each amplified by PCR (see FIG. 2). ). The PCR primers used were the former TB604 (5'-TTCGCATCTAAGGGGTGGTG-3 ': SEQ ID NO: 11) and TB607 (5'-GCGTGTACGCATGTAACTTTGTTCTTCTTGTTATT-3': SEQ ID NO: 12), and the latter TB599 (5'-CTAACATTCAACGCTAGACGGTTCTCTACAATTGA-3 ': SEQ ID NO: 13) and TB501 (5′-TAAGAAGGCATGTTGGCCTCTGT-3 ′: SEQ ID NO: 14).

In addition, the hygromycin resistance gene expression cassette was amplified by PCR using the plasmid pBHPH-PT disclosed in JP-A-2003-259878 as a template. The PCR primers used were TB606 (5′-AATAACAAGAAGAACAAAGTTACATGCGTACACGC-3 ′: SEQ ID NO: 15) and TB598 (5′-TCAATTGTAGAGAACCGTCTAGCGTTGAATGTTAG-3 ′: SEQ ID NO: 16).

Using the three types of PCR products amplified as described above as a template, the three types of PCR products were combined by the method of Shevchuk, NA, et al. In this PCR, TB070 (5′-GGAGACCCACTGTACAAC-3 ′: SEQ ID NO: 17) and TB210 (5′-GCAGCTGAAAGATAATAAGGTATG-3 ′: SEQ ID NO: 18) were used as primers. Hereinafter, this DNA fragment is referred to as “PDC5 gene disruption DNA fragment”.

Preparation of DNA fragment for overexpression of HAP4 gene In this example, a DNA fragment was prepared in order to overexpress the HAP4 gene derived from Saccharomyces cerevisiae. Specifically, using the genomic DNA of Saccharomyces cerevisiae BY4742 as a template, a DNA fragment containing a part of the PDC6 gene and its 5 ′ upstream untranslated region, a DNA fragment containing the HAP4 gene and its terminator region, and a TDH2 promoter region The DNA fragment and the DNA fragment containing the 5 ′ upstream untranslated region of the CTT1 gene were amplified by PCR (see FIG. 3). The PCR primers used were TB1021 (5'-CCTTGATGCGTGCGTAACC-3 ': SEQ ID NO: 19) and TB910 (5'-AAACGCGTGTACGCATGTAATCTCATAAACCTATGCACTG-3': SEQ ID NO: 20), TB911 (5'-TCATATTCGACGATGTCGTCCGAACTACAGTTATCGCCTC-3 ': SEQ ID NO: 21 ) And TB679 (5'-CACACAAACAAACAAAACAAAATGACCGCAAAGACTTTTCTAC-3 ': SEQ ID NO: 22), TB680 (5'-GTAGAAAAGTCTTTGCGGTCATTTTGTTTTGTTTGTTTGTGTG-3': SEQ ID NO: 23) and TB900 (5'-ATATATCTGCAGGGATCCCTTGAC899) '-TCAGAATACCCGTCAAGGGATCCCTGCAGATATAT-3': SEQ ID NO: 25) and TB349 (5'-CCATATTTTCGTTAGGTCATTT-3 ': SEQ ID NO: 26).

Further, the phleomycin expression cassette was amplified by PCR using the plasmid pBble-LDHKCB disclosed in JP-A-2003-259878 as a template. The PCR primers used were TB909 (5′-CAGTGCATAGGTTTATGAGATTACATGCGTACACGCGTTT-3 ′: SEQ ID NO: 27) and TB912 (5′-GAGGCGATAACTGTAGTTCGGACGACATCGTCGAATATGA-3 ′: SEQ ID NO: 28).

A PCR product containing a part of the PDC6 gene amplified as described above and its 5 ′ upstream untranslated region and a phleomycin expression cassette as a template were combined by PCR according to the method of Shevchuk, NA, et al. The PCR primers used were TB315 (5′-ACCAGCCCATCTCAATCCATCT-3 ′: SEQ ID NO: 29) and TB912. The PCR product containing the HAP4 gene amplified as described above and its terminator region, the PCR product containing the TDH2 promoter region, and the PCR product containing the 5 'upstream untranslated region of the CTT1 gene were similarly combined by PCR. . The PCR primers used were TB911 and TB316 (5′-AGCGTATGGGTGATGAGAGTAC-3 ′: SEQ ID NO: 30).

The two fragments combined as described above were further combined by PCR in the same manner using DNA as a template. In this PCR, TB948 (5′-GTTGAAGTCGCCTGGTAGCC-3 ′: SEQ ID NO: 31) and TB734 (5′-TGTCCAGGCTACGTCGAATC-3 ′: SEQ ID NO: 32) were used as primers. The finally obtained DNA fragment is referred to as “HAP4 gene overexpression DNA fragment”.

LDH gene transfer / PDC1 gene disruption strain production Using the Frozen-EZ Yeast Transformation II kit (ZYMO RESEARCH), the above-mentioned DNA fragment for LDH gene transfer / PDC1 gene disruption is introduced into BY4742 strain for criminal conversion. went. This transformation followed the protocol attached to the kit. After transformation, the cells were spread on a leucine selective medium (SD-Leu) plate and cultured at 30 ° C. for 3 days to select transformants. Genomic DNA was prepared from the transformant, and it was confirmed by PCR that the DNA fragment for LDH gene introduction / PDC1 gene disruption was integrated into the chromosome. In this PCR, TB324 (5′-CTCATACATGTTTCATGAGGGT-3 ′: SEQ ID NO: 33) and TB304 (5′-ACACCCAATCTTTCACCCATCA-3 ′: SEQ ID NO: 34) are used as primers located outside the DNA fragment for LDH gene transfer / PDC1 gene disruption. It was used. As a result, the PDC1 gene in BY4742 strain was destroyed, and it was confirmed that the LDH gene was integrated into the chromosome. Hereinafter, this transformed yeast is referred to as “LDH gene introduced / PDC1 gene disrupted strain”.

LDH gene introduction / PDC1 and PDC5 gene disruption strain production Next, the LDH gene introduction / PDC1 gene disruption strain was transformed using the PDC5 gene disruption DNA fragment. The transformation method is the same as described above. After transformation, the cells were plated on a plate of YPD medium containing 200 μg / ml hygromycin, cultured at 30 ° C. for 3 days, and transformants were selected. Genomic DNA was prepared from the transformant, and it was confirmed by PCR that the DNA fragment for PDC5 gene disruption was integrated into the chromosome. In this PCR, TB077 (5′-GGAACCCATAGATGAAGAGG-3 ′: SEQ ID NO: 35) is used as a primer located outside the DNA fragment for PDC5 gene disruption, and TB434 (5′-ATCCGCTCTAACCGAAAAGG-) is used as a primer located inside the DNA fragment for PDC5 gene disruption. 3 ′: SEQ ID NO: 36) was used. As a result, it was confirmed that the PDC5 gene was disrupted in the LDH gene-introduced / PDC1 gene-disrupted strain. Hereinafter, this transformed yeast is referred to as “LDH gene introduced / PDC1 and PDC5 gene disrupted strain”.

Preparation of HAP4 gene-introduced strain Next, the above-mentioned DNA fragment for HAP4 gene overexpression was transformed into the LDH gene-introduced / PDC1 gene-disrupted strain and the LDH gene-introduced / PDC1 and PDC5 gene-disrupted strain. The transformation method is the same as described above. After transformation, the cells were spread on a plate of YPD medium containing 100 μg / ml phleomycin and cultured at 30 ° C. for 3-5 days to select transformants. Genomic DNA was prepared from the transformant, and it was confirmed by PCR that the DNA fragment for HAP4 gene overexpression was integrated into the chromosome. This PCR uses TB315, a primer located outside the HAP4 gene overexpression DNA fragment, and TB1020 (5'-TCCTGCGCCTGATACAGAAC-3 ': SEQ ID NO: 37) located inside the HAP4 gene overexpression DNA fragment. did. As a result, it was confirmed that the DNA fragment for overexpression of the HAP4 gene was introduced into the chromosomes of the LDH gene introduced / PDC1 gene disrupted strain and the LDH gene introduced / PDC1 and PDC5 gene disrupted strain.

Proliferation test LDH gene-introduced / PDC1 gene-disrupted strain (in this section, HAP4 non-introduced strain) and LDH gene-introduced / PDC1 gene-disrupted strain (in this section, HAP4 introduced) The specific growth rate was calculated for the strain. Specifically, the test strain was inoculated into a 500 ml baffled flask in which 100 ml of YPD (yeast extract 1%, peptone 2% and glucose 2%) liquid medium was dispensed, and 30 ° C, 120 rpm (amplitude 35 mm) After 15 to 20 hours of shaking culture, the cells were collected at a cell concentration of 0.7 to 1.0%. Again, the test strain was inoculated at the same concentration of 0.01% under the same conditions, and sampling was performed approximately every 2 hours after the start of growth to measure the bacterial cell concentration. The specific growth rate was confirmed to be in the logarithmic growth phase for 2 to 10 hours from the start of growth, and calculated according to the following formula.

The results of the proliferation test are shown in Table 1. As shown in Table 1, the HAP4-introduced strain had almost the same growth rate as the HAP4-non-introduced strain.

Fermentation test 1
LDH gene-introduced / PDC1 gene-disrupted strains prepared in the manner described above (HAP4 non-introduced strain in this section) and LDH gene-introduced / PDC1 gene-disrupted strains into which HAP4 gene overexpression DNA fragments have been introduced (in this section, HAP4-introduced strains) A fermentation test was conducted. Specifically, the test strain was inoculated into a YPD medium (yeast extract 1%, peptone 2% and glucose 2%) and cultured at 30 ° C. for 24 hours. After completion of the culture, the cells were collected by centrifugation (2000 g, 3 minutes).

Next, 25 ml of fermentation medium (11% glucose, 1% yeast extract and 4% calcium carbonate) is placed in a 50 ml flask and inoculated so that the bacterial cell concentration is 0.5%. 80 rpm / min (shake width 40 mm) ) At 34 ° C. for 2 to 3 days. After the fermentation was finished, production amounts of lactic acid and ethanol were examined. The lactic acid yield was calculated by the following formula.

Lactic acid yield (%) = Lactic acid maximum concentration (%) / Preparation sugar concentration (%)
The results of the fermentation test are shown in FIG. As can be seen from FIG. 4, in the HAP4-introduced strain, the lactic acid yield was improved from 62% to 70% and the maximum ethanol concentration was decreased from 2.2% to 1.4%, compared with the non-HAP4-introduced strain. In addition, the fermentation rate was comparable.

From this result, it was clarified that in the yeast that constitutively expressed the HAP4 gene, the ability to produce lactic acid by the LDH gene introduced as a foreign gene was greatly improved.

Fermentation test 2
LDH gene-introduced / PDC1 and PDC5 gene-disrupted strains (in this section, HAP4-non-introduced strains) and LDH gene-introduced / HAP4 gene overexpression DNA fragments / PDC1 and PDC5 gene-disrupted strains (in this section) A fermentation test was conducted on HAP4-introduced strains. This fermentation test was performed in the same manner as in the fermentation test 1 except that a YPE medium (1% yeast extract, 2% peptone, and 1% ethanol) was used instead of the YPD medium.

The results of the fermentation test are shown in FIG. As can be seen from FIG. 5, in the HAP4-introduced strain, the lactic acid yield was improved from 77% to 90% and the maximum ethanol concentration was decreased from 0.09% to 0.03% compared to the non-HAP4-introduced strain. The fermentation rate was about the same.

From this result, it was clarified that in the yeast that constitutively expressed the HAP4 gene, the ability to produce lactic acid by the LDH gene introduced as a foreign gene was greatly improved. In addition, compared with the results of fermentation test 1 (FIG. 4), yeast that constitutively expresses the HAP4 gene and has a lower alcohol productivity has a higher ability to produce lactic acid by the LDH gene introduced as a foreign gene. It became clear that it improved more greatly.

All publications, patents and patent applications cited in this specification shall be incorporated into the present specification as they are.

Claims (13)

1. A mutant yeast into which a foreign gene encoding an enzyme involved in the production of a target product and a constitutively expressed HAP4 gene or a homologous gene thereof are introduced.
2. The mutant yeast according to claim 1, wherein the mutant yeast has a reduced alcohol productivity as compared with the wild type.
3. 3. The mutant yeast according to claim 1 or 2, wherein the enzyme activity of an enzyme involved in alcohol synthesis is reduced.
4. The mutant yeast according to claim 3, wherein the enzyme involved in alcohol synthesis is pyruvate decarboxylase and / or alcohol dehydrogenase.
5. The mutant yeast according to claim 4, wherein the pyruvate decarboxylase is encoded by at least one gene selected from the group consisting of PDC1 gene, PDC5 gene and PDC6 gene.
6. The mutant yeast according to claim 4, wherein the alcohol dehydrogenase is encoded by an ADH1 gene.
7. The mutant yeast according to any one of claims 1 to 6, which belongs to the genus Saccharomyces.
8. The mutant yeast according to any one of claims 1 to 6, which belongs to a strain of Saccharomyces cerevisiae.
9. The mutant yeast according to any one of claims 1 to 8, wherein the foreign gene is a gene encoding a protein having lactate dehydrogenase activity.
10. Culturing the mutant yeast according to any one of claims 1 to 8, and producing a target product inside and outside the fungus body;
    A method for producing a substance using yeast, comprising the step of recovering the target product.
11. 11. The substance production method according to claim 10, wherein the target product is an organic acid.
12. 11. The method for producing a substance according to claim 10, wherein the target product is lactic acid.
13. 11. The substance production method according to claim 10, wherein the target product is an alcohol other than ethanol.

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